JPH10245377A - Novel alkaloids ulbactin D and E, and methods for their production - Google Patents

Abstract

(57) [Summary] [Solution] The following formula (I) or (II): Embedded image And a method for producing the compound using a microorganism. [Effect] A substance useful as an ultraviolet absorber is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel compound useful as an ultraviolet absorbing material, and a method for producing the same using a microorganism.

[0002]

2. Description of the Related Art Conventionally, benzophenone derivatives, paraaminobenzoic acid derivatives,
Chemically synthesized products such as paramethoxycinnamic acid derivatives and salicylic acid derivatives have been mainly used. However, with the rejection of chemically synthesized products, research is being conducted to search for novel UV-absorbing substances having high safety from natural products, and to apply and utilize them. Mycosporine-like amino acids produced by seaweeds, marine animals, microalgae, molds, etc. are known as natural ultraviolet absorbing substances (Botanica Marina (Bot. Mar.) 16, 23 (1974), Marine Biology (Mar. Biol.) 108, 157 (1991), J. Plankton Res. 12, 909.
(1990), Plant Diss Reporter
eptr.) 57,760 (1973)).

[0003]

However, since these substances are water-soluble, their application range is limited. For this reason, a fat-soluble ultraviolet absorbing substance derived from a natural product has been demanded. The present invention has been made under such a technical background, and an object of the present invention is to provide a novel ultraviolet absorbing substance and a method for producing the same.

[0004]

Means for Solving the Problems The present inventors have found that a compound obtained from a culture of a strain belonging to the genus Alteromonas is a novel compound that has never been known before, and completed the present invention. That is, the present invention provides the following formula (I) or (II)

[0005]

Embedded image

[0006]

Embedded image

Is a compound represented by the formula:

[0008] The present invention also relates to a microorganism belonging to the genus Alteromonas, which is capable of producing the above-mentioned compound, is cultured in a medium, and the above-mentioned compound is produced and accumulated in the culture. A method for producing the compound described above, wherein the compound is collected.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. (1) Properties of the compound of the present invention A group of novel compounds of the present invention is represented by the following formula (I) or (I)
It is represented by I).

[0010]

Embedded image

[0011]

Embedded image

[0012] The present inventors have, among these novel compounds,
The compound represented by the formula (I) is referred to as “Ulbactin D (Ulbac
tin D) "and the compound represented by the formula (II) were designated as" Ulbactin E ". The physicochemical properties of each compound are shown below.

<Urbactin D> (1) Color of substance: colorless (2) Shape of substance: oily (3) Molecular weight: 379 (4) Molecular formula: C ₁₆ H ₁₇ N ₃ O ₂ S ₃ (5) Mass spectrometry: High resolution FABMS actual value 380.0566
[M + H] ⁺ calculated value 380.0561 (C ₁₆ H ₁₈ N ₃ O ₂ S ₃ ) (6) Specific rotation: [α] _D ²⁵ + 257 ° (c 0.0033 in chloroform) (7) Ultraviolet absorption spectrum (in methanol) ): Λ _max 316 nm (log ε3.68), 253 (4.0
9), 212 (4.44) (8) Infrared absorption spectrum (KBr) ν _max 3442, 2934, 1659, 1622, 1597, 1491, 1230 cm
^-1

(9) ¹ H-NMR (measured in deuterated chloroform, 400 MHz) δ ppm 2.91 (dd, J = 9.6, 4.8 Hz 1H), 3.03 (dd, J = 1
1.2, 9.6Hz, 1H), 3.13 (dd, J = 10.7, 6.4Hz, 1H), 3.
28 (dd, J = 11.2, 8.0Hz, 1H), 3.36 (dd, J = 11.2, 8.0H
z, 1H), 3.50 (d, J = 10.7Hz, 1H), 3.97 (dd, J = 11.2,
4.8Hz, 1H), 4.50 (d, J = 6.4Hz, 1H), 4.90 (qd, J =
8.0Hz, 1H), 5.14 (s, 1H), 5.62 (d, J = 8.0Hz, 1H
H), 6.86 (ddd, J = 7.8, 7.6, 1.2Hz, 1H), 6.99 (dd,
J = 8.4, 1.2Hz, 1H) 7.34 (ddd, J = 8.4, 7.6, 1.6Hz, 1H), 7.39 (dd, J = 7.
(8, 1.6Hz, 1H)

(10) ¹³ C-NMR (measured in deuterated chloroform, 125 MHz) δ ppm 34.1 (t), 34.3 (t), 40.4 (t), 61.3 (d), 65.
1 (d), 65.3 (d), 69.2 (d), 81.0 (d), 116.2 (s), 117.
2 (d), 118.9 (d), 130.7 (d), 133.4 (d), 159.2 (s),
167.9 (s), 174.2 (s) (11) Solubility: Soluble in methanol, ethanol, acetone, ethyl acetate and chloroform, poorly soluble in water (12) Color reaction: positive coloring of ferric chloride

(13) Thin layer chromatography: Silica gel thin layer (Kieselgel 60 F 254 Art.5554. Merck) Chloroform: Methanol (10: 1) Rf value in developing solvent is 0.48 (14) High performance liquid chromatography : Develosil packed column ODS-HG-5 4.6 mmφ, 25 cm, manufactured by Nomura Chemical Co., Ltd., acetonitrile: water (0 to 5 minutes: 20% acetonitrile, 5 to 6)
Min: 20-36% acetonitrile, 6-35 min: 36%
Acetonitrile, 35 to 36 minutes: 36 to 60% acetonitrile, 36 to 50 minutes: 60% acetonitrile), flow rate
The retention time at 1.0 ml / min is 44.8 minutes.

<Ulbactin E> (1) Color of substance: yellow (2) Shape of substance: oily (3) Molecular weight: 264 (4) Molecular formula: C ₁₂ H ₁₂ N ₂ OS ₂ (5) Mass spectrometry: High resolution FABMS actual value 265.0489
[M + H] ⁺ calculated value 265.0469 (C ₁₂ H ₁₃ N ₂ OS ₂ ) (6) Specific rotation: [α] _D ²⁴ -19 ° (c 0.015 in chloroform) (7) Ultraviolet absorption spectrum (in methanol): λ _max 315 nm (log ε3.72), 252 (4.1
(0), 213 (4.41) (8) Infrared absorption spectrum (KBr) ν _max 3446, 1622, 1595, 1491, 1255, 1222 cm ^-1

(9) ¹ H-NMR (measured in deuterated chloroform, 500 MHz) δ ppm 3.27 (dd, J = 10.8, 9.0 Hz, 1H), 3.30 (dd, J =
10.8, 9.0Hz, 1H), 3.98 (dd, J = 4.3, 1.3Hz, 2H), 5.
38 (td, J = 9.0, 4.0Hz, 1H), 6.37 (tdd, J = 4.3, 4.0,
2.4Hz, 1H), 6.87 (td, J = 7.6, 1.2Hz, 1H), 6.99 (d
d, J = 8.2, 1.2Hz, 1H), 7.35 (ddd, J = 8.2, 7.6, 1.6H
z, 1H), 7.39 (dd, J = 7.6, 1.6Hz, 1H) 7.58 (dt, J = 2.4, 1.3Hz, 1H)

(10) ¹³ C-NMR (measured in deuterated chloroform, 125 MHz) δ ppm 32.0 (t), 44.6 (t), 80.3 (d), 87.3 (d), 11
6.1 (s), 117.1 (d), 118.8 (d), 130.7 (d), 133.2
(d), 159.2 (s), 162.5 (d), 173.2 (s) (11) Solubility: soluble in methanol, ethanol, acetone, ethyl acetate and chloroform, poorly soluble in water (12) Color reaction: Ferric chloride coloring positive

(13) Thin layer chromatography: silica gel thin layer (Kieselgel 60 F 254 Art.5554, Merck) Chloroform: methanol (10: 1) Rf value in developing solvent is 0.63 (14) High performance liquid chromatography : Develosil packed column ODS-HG-5 4.6 mmφ, 25 cm, manufactured by Nomura Chemical Co., Ltd., acetonitrile: water (0 to 5 minutes: 20% acetonitrile, 5 to 6)
Min: 20-36% acetonitrile, 6-35 min: 36%
Acetonitrile, 35 to 36 minutes: 36 to 60% acetonitrile, 36 to 50 minutes: 60% acetonitrile), flow rate
The retention time at 1.0 ml / min is 43.5 minutes.

(2) Production of the Compound of the Present Invention The compound of the present invention is obtained by culturing a microorganism in a medium, producing and accumulating the compound of the present invention in a culture, and collecting the compound of the present invention from the culture. Can be manufactured. The microorganism used here belongs to the genus Alteromonas,
There is no particular limitation as long as it has the ability to produce the compound of the present invention, and examples thereof include Alteromonas sp. Strain D-12. Also, a mutant of the strain,
Strains capable of producing the compounds of the invention can also be used.

Alteromonas sp. Strain D-12 is a strain newly isolated from nature and its bacteriological properties are as follows. The morphological observation was performed using an optical microscope and a transmission electron microscope after inoculating Alteromonas sp. D-12 strain in the medium and culturing the cells at 30 ° C. for 3 to 5 days. The medium was marine agar (Bacto Marine Agar 2216,
Difco) or marine broth (Bacto Marine Brot)
h 2216, Difco). Since the bacterial growth of this strain is not observed when the concentration of sodium chloride in the culture medium is 1% or less, the biochemical properties were tested by preparing the culture medium using 75% artificial seawater, or Performed by adding 3% sodium chloride.

A. Morphology 1) Cell shape and size: Bacillus, 1.6-2.5 × 0.8-1.1
μm 2) Cell polymorphism: No 3) Motility, Flagellar status: Yes, polar hair. 4) Spore presence: none 5) Gram stainability: negative 6) Acid resistance: none

B. Growth state in each medium 1) Marine agar plate culture: good growth, colony is circular, convex, whole edge, wet light, yellow 2) Marine agar slope culture: good growth, filament, yellow 3) Marine broth culture: Good growth, film formation, and ring formation, and the upper part of the medium becomes turbid. 4) Marine broth gelatin puncture culture: liquefy. 5) Litmus milk: Digest.

C. Physiological properties 1) Reduction of nitrate: not reduced. 2) MR test: negative 3) VP reaction: negative 4) Indole production: no production. 5) Generation of hydrogen sulfide: No generation. 6) Hydrolysis of starch: Decomposes. 7) Use of citric acid: Simmons medium: Not used. : Cristensen medium: Use.

8) Utilization of inorganic nitrogen source: Utilization. 9) Pigment production: Produces water-insoluble yellow pigment. 10) Urease activity: Negative 11) Oxidase activity: Positive 12) Catalase activity: Positive 13) Growth range (Temperature, pH): Best growth at temperature 12-41 ℃, 30-38 ℃ pH3-10, Optimal growth pH range 5-10 14) Attitude to oxygen: aerobic 15) OF test: degrades glucose oxidatively.

16) Presence or absence of generation of acid and gas from saccharides Use Baumann's BM medium as a basal medium After culturing for 3 weeks at 25 ° C. Generation of acid Generation of gas L-arabinose-D-xylose-D-glucose + -D-mannose +-D-fructose +-D-galactose--maltose +-sucrose +-lactose--trehalose +-D-sorbitol--D-mannitol--inosit--glycerin--starch +-- Not generated + Generated 17) Esculin decomposition: Not decomposed. 18) Decomposition of arginine: Does not decompose. 19) DNA degradation: Decomposes. 20) Halophilicity: Yes Growth salt concentration range 2-12% 21) β-galactosidase: negative 22) GC content (mol%): 46.9 23) Isoprenoid quinone: ubiquinone Q-8

The above mycological properties were compared with known bacterial species according to the classification criteria of the Virgie's Manual of Systematic Bacteriology, Eighth Edition. This strain was an aerobic gram-negative bacillus with polar flagella, oxidatively degraded glucose, and was positive for oxidase and catalase. Furthermore, liquefied gelatin, the DNA was hydrolyzed, believed to GC content belongs to genus Alteromonas from 50% or less, Alteromonas species (Alteromo
nas sp.). Alteromonas sp. Strain D-12 is
It has been deposited as FERM P-15733 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology on July 12, 1996.

For culturing the microorganism used in the present invention, a usual method for culturing a microorganism is used. As the medium, any of a synthetic medium and a natural medium can be used as long as the medium contains assimilable carbon sources, nitrogen sources, inorganic substances, and necessary growth and production promoting substances. As a carbon source, glucose, starch, dextrin, mannose, fructose,
Sucrose, lactose, xylose, arabinose, mannitol, molasses and the like are used alone or in combination. Furthermore, depending on the assimilation ability of bacteria, hydrocarbons,
Alcohols, organic acids and the like are also used. As the nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean powder, casamino acid and the like are used alone or in combination. In addition, if necessary, salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate,
Add inorganic salts such as dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate and zinc sulfate. Further, a trace component that promotes the growth of the bacterium used and the production of ulbactin D and ulbactin E can be appropriately added. The most suitable culture method is a liquid culture method. A culture temperature of 30 to 35 ° C. is appropriate, and the pH of the medium during the culture is
Is preferably maintained at 7 to 8. Usually 3 in liquid culture
After culturing for up to 5 days, the target substance is produced and accumulated in the culture solution. The culture is stopped when the production in the culture reaches a maximum.

The isolation and purification of ulbactin D and ulbactin E from a culture are performed according to a method commonly used for isolating and purifying a microbial metabolite from the culture. For example, the culture is separated into a culture filtrate and cells by filtration or centrifugation, and the cells are extracted with aqueous methanol, aqueous ethanol, or the like. Then, the extract and the culture filtrate are combined and concentrated, and ulbactin D and ulbactin E are obtained by column chromatography using silica gel, gel filtration, high performance liquid chromatography, or the like. In addition, culture,
The trends of ulbactin D and ulbactin E during the purification operation can be tracked using ultraviolet absorption by high performance liquid chromatography as an index.

(3) Use of the compound of the present invention The compound of the present invention has a strong ability to absorb ultraviolet light, and thus can be used as an ultraviolet absorbing component in cosmetics and the like. Hereinafter, the present invention will be described specifically with reference to Examples. However, the technical scope of the present invention is not limited by these examples.

[0032]

EXAMPLES As the inoculum, Alteromonas sp. Strain D-12 was used. The strain was added to 50 ml of kelp tea liquid medium (kelp tea: 1).
0 g, glucose: 3.0 g, broth: 1.0 g, calcium carbonate: 2.0 g, filtered seawater: 500 ml, distilled water: 500 ml, pH = 7.4). After culturing for 100 hours with shaking (100 rpm), a pre-pre-culture solution was obtained. During the culture, the pH of the medium was not particularly controlled. 12 ml of the pre-pre-culture liquid was inoculated into a 1 L Erlenmeyer flask containing 300 ml of kelp tea liquid medium,
Culturing was performed at 100 ° C. for 8 hours with shaking (100 rpm) to obtain a pre-culture solution. The whole amount of the preculture solution was inoculated into a 5 L jar fermenter containing 3 L of kelp tea liquid medium, and was inoculated at 30 ° C. and 200 rp.
Main culture was performed for 5 days at a flow rate of 0.2 L / min.

3.3 L of the culture thus obtained was centrifuged, and the resulting supernatant was washed with ethyl acetate (6.6).
L). The ethyl acetate extract was concentrated under reduced pressure at 40 ° C or lower, and the resulting concentrated solution was subjected to reverse phase high performance liquid chromatography (Develosil packed column ODS-HG-5 20 mmφ, 25 cm
Nomura Chemical Co., Ltd., water: acetonitrile = 80: 20-4
0:60) to give two fractions. The first fraction is a silica gel column chromatography (Sep-Pak (registered trademark) Vac 12cc, manufactured by Waters,
Purification by hexane-acetone = 10: 1) yielded 0.6 mg of ulbactin D. The second fraction was a silica gel column chromatography (Sep-Pak® Vac 12cc,
Purification was performed using Waters, hexane-ethyl acetate = 10: 1) to obtain 0.8 mg of ulbactin E. The ulbactin D and ulbactin E obtained here exhibited the above-mentioned physicochemical properties.

[0034]

The present invention provides a novel compound and a method for producing the substance using a microorganism. The compounds of the present invention
Since it is fat-soluble and has a strong ability to absorb ultraviolet light, it can be used as a component of cosmetics.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:01) (C12P 17/18 C12R 1:01) (72) Inventor Miyuki Nishijima 1900 Sodeso-cho, Shimizu-shi, Shizuoka Co., Ltd. Kaiyo Biotechnology Research Laboratories Shimizu Research Institute (72) Inventor Fumiko Nishida 1900 Sodesoshi-cho, Shimizu City, Shizuoka Prefecture Kaiyo Biotechnology Research Laboratories Shimizu Research Institute (72) Inventor Takahide Takadera 1900 Sodesomicho, Shimizu-shi Shizuoka Prefecture Kaiyo Biotechnology Research Institute, Inc. Shimizu Research Institute (72) Inventor Hiroshi Sano 1900 Sodesoshi-cho, Shimizu City, Shizuoka Prefecture Kaiyo Biotechnology Research Institute, Shimizu Research Institute

Claims (2)

[Claims] 1. A compound represented by the following formula (I) or (II): Embedded image A compound represented by the formula: 2. A microorganism belonging to the genus Alteromonas and having the ability to produce the compound according to claim 1 is cultured in a medium, and the compound according to claim 1 is produced and accumulated in the culture.
The method for producing a compound according to claim 1, wherein the compound according to claim 1 is collected from the culture.